1. Field of the Invention
The present invention relates to a method for producing cytosine nucleoside compounds, which are useful as starting material for synthesis of compounds, such as those for medical use. More specifically, the invention relates to a method for producing cytosine nucleoside compounds from pentose-1-phosphate and cytosine or a cytosine derivative, by using an enzyme with cytosine-nucleoside phosphorylase activity, microbial cells with the enzyme activity, an enzyme preparation from the microbial cells or the culture thereof, or the like.
2. Description of the Related Art
Nucleoside phosphorylases generally refer to enzymes which phosphorolyze the N-glycoside bond of a nucleoside in the presence of phosphoric acid, and in the case where ribonucleoside is used, catalyze a reaction represented by the following equation:ribonucleoside+phosphoric acid (or a salt thereof)→base for nucleic acid+ribose-1-phosphate. 
Those enzymes, roughly divided into purine-nucleoside phosphorylases and pyrimidine-nucleoside phosphorylases, are distributed in a wide variety of living organisms, such as tissues including those of mammals, birds or fish, yeast or bacteria. These enzyme reactions are reversible, and synthesis of different nucleosides thus utilizing the reverse rections has been known. For example, there are known methods for production of thymidine (Japanese Patent Laid-Open No. 01-104190), 2′-deoxyadenosine (Japanese Patent Laid-Open No. 11-137290) or 2′-deoxyguanosine (Japanese Patent Laid-Open No. 11-137290), respectively, from 2′-deoxyribose-1-phosphate and a base for nucleic acid (thymine, adenine or guanine). Thus, production of a nucleoside using a phosphorylase can be produced regio-specifically and stereospecifically in a mild condition, and many nucleoside compounds are studied for synthesis.
Japanese Patent Laid-Open No. 1-60396 describes a process for production of deoxycytidine by the reaction of deoxyribose-1-phosphate with cytosine using bacterial cells themselves as catalyst. Though the process of the application uses bacterial cells per se as reaction catalyst, it is uncertain whether cytosine-nucleoside phosphorylase itself may exist therein. There is a possibility such that deoxycytidine accumulated in the bacterial cells may have leached out of the cells during the reaction, or that nucleoside deoxyribosyltransferase within the cells may have transferred cytosine added as substrate to the base of deoxynucleoside within the cells, resulting in detection of deoxycytidine. The inventors sent for the seven bacterial strains that have been deposited for the examples of the application, and tested the production of deoxycytidine from deoxyribose-1-phosphate and cytosine according to the examples. As a result, deoxycytidine could not be detected in the solution of the reaction using any one of the strains, indicating that enzyme activity corresponding to cytosine-nucleoside phosphorylase was not present in the strains themselves.
On the other hand, Japanese Patent Laid-Open No. 3-127986 describes a novel nucleoside phosphorylase acting on both purine and pyrimidine bases, where the pyrimidine base is deoxyuridine, deoxycytidine or deoxythymidine, but no substrate specificity of the enzyme is disclosed in the specification. Further, the application has already been withdrawn and therefore the enzyme activity cannot be characterized any more. The inventors of the application, however, purified a nucleoside phosphorylase from the strain of the same designation as that disclosed in the application, and reported the characteristics thereof in Applied and Environmental Microbiology, Vol. 56, pp. 3830-3834 (1990). The article states that the above enzyme has no activity to cytosine, cytidine of deoxycytidine.
Apart from the enzyme described above, it is not known that cytosine or any derivative thereof may be a substrate for either purine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase. For example, thymidine nucleoside phosphorylase, one of pyrimidine nucleoside phosphorylases, derived from Salmonera typhimurium described in Method. Enzymology, Vol. 51, pp. 437-442 (1978) has no activity to deoxycytidine. In addition, it is reported in J. Biol. Chem., Vol. 248, No. 6, pp. 2040-2043 (1973) that purine nucleoside phosphorylase derived from Salmonera typhimurium has no activity to pyrimidine nucleoside, such as uridine, cytidine, deoxyuridine or deoxycytidine.
When these publications are totally considered, an enzyme corresponding to cytosine nucleoside phosphorylase related to the present invention may have been existent, but none of those preceding publications could confirm it and actually obtain the enzyme.